The objectives of this proposal are to determine a) the factors and mechanisms that modulate biofilm formation and dispersal as a function of cellular respiration in Staphylococcus aureus, b) determine how the expression of these factors is regulated, and c) determine the molecular stimuli for a regulatory system that responds to alterations in the cellular respiratory status. Staphylococcus aureus is a human commensal and a leading cause of hospital and community acquired infections. For S. aureus to colonize and invade host tissues, it must successfully sense and respond to environmental perturbations and transition between individualistic and multicellular behaviors. S. aureus has the ability to form multicellular communities called biofilms, which are the etiologic agents of recurrent staphylococcal infections. We have found that S. aureus modulates biofilm formation as a function of electron flux though respiratory pathways. We have found that when S. aureus is growing without a terminal electron acceptor (fermentative growth) there is an increase in cell lysis, which leads to the release of intracellular components including DNA. The DNA that is released is vital to biofilm integrity where it aids in holding the cells together in the biofilm matrix. We also discovered that the addition of a terminal electron acceptor to fermentative biofilms resulted in biofilm dispersal. We have identified two global regulatory systems that mediate fermentative biofilm formation. Both of these regulatory systems have been shown to be necessary for pathogenesis, but their molecular stimuli are currently unknown. The overarching goal of this project is to understand the physiological changes that occur within S. aureus upon changes in cellular respiration, and examine how these changes affect the community structure. We will use genetic, physiologic, biochemical, and molecular techniques to investigate an understudied variable that causes S. aureus to modulate cell lysis, and thereby affect biofilm formation and biofilm dispersal. We will further define the factors involved in fermentative cell lysis and biofilm formation. We will also define the factors and molecular mechanisms involved in the dispersal of fermentative biofilms. We will examine the molecular stimuli of one regulatory system (Sae) and examine how another regulatory system (Srr) controls the expression of factors that alter cell lysis and biofilm formation. Ultimately, we would like to apply our findings to modulate the behavior of S. aureus using molecules that alter cell signaling, and thereby, positively affect infection outcomes. Completion of the studies will help provide knowledge about two requirements for staphylococcal pathogenesis: environmental sensing and biofilm formation.